New approach to enhance cell-free biosynthesis with engineered yeast extracts

Think of cells as tiny factories: within their walls they have both the machinery to make products such as proteins, and the mechanisms for maintenance to keep processes running smoothly.

As reported August 26, 2021, in Nature Communications, researchers led by Hal Alper at The University of Texas at Austin and Michael Jewett of Northwestern University describe a two-pronged approach that starts with engineered yeast cells but then moves out of the cell structure into a cell-free system. The work complements efforts to further develop sustainable alternative approaches for manufacturing bioproducts and biofuels. This first report of their work, supported through the Emerging Technologies Opportunity Program at the U.S. Department of Energy (DOE) Joint Genome Institute (JGI), a DOE Office of Science User Facility located at Lawrence Berkeley National Laboratory, indicates metabolic pathways could be more productive outside the cell environment, which would be transformative at industrial scales.

Cell-free systems have been really powerful at finding ways to accelerate bio-design and identifying the best sets of enzymes that enable synthesis of sustainable chemical products. But this has been done most commonly in bacterial-based systems, and so one of the things that I think is so special about this collaboration is that we're doing work in yeast, a key platform organism that has been used historically for making sustainable products in industry."

Michael Jewett, Study's Senior Author, Northwestern University

From cell to "soup"

The work started with yeast strains engineered by Alper's team to convert the sugar glucose to the products including ethanol and 2,3-butanediol (BDO). To do so, the yeasts received fine-tuning of gene expression through genetic rewiring with CRISPR-Cas9 that enhanced their ability to redirect the carbon in the cells toward particular pathways. In this case, towards BDO and away from ethanol, an undesired product of the yeast's native glucose metabolism.

Alper's graduate student Xiunan Yi visited the Jewett lab to provide a seamless transition between the in vivo (within the cell)and in vitro (outside the cell) efforts of this work when it started. Though such visits have been impossible for the past year and a half, "the logistics haven't changed," noted Northwestern graduate student Blake Rasor. He and Yi, one of the study co-authors, have maintained a regular shipment schedule, with plates arriving every couple of months.

The question for the researchers was, would the reactions in these engineered yeast strains continue to work in a cell-free system? At the Jewett lab, the engineered cells were lysed or burst, and the metabolic pathways were tested in the enhanced lysate "soup." In this cell-free environment, reported Rasor, first author of the study, the lysate produced BDO at higher productivity rates than were noted inside the cell.

"One of the huge advantages of the cell-free system is that the cells are no longer growing," Rasor said. "So they can push more of the carbon that we feed into a product molecule and so we're able to make the same amount of a product in just eight hours that growing cells take three days to make, which is a really exciting jump in the rate of product formation."

This was the first time CRISPR-Cas9 had been used to modify a cell's genome in order to create lysates with altered metabolic performance, Alper noted. "This was really an unknown exercise," he added. "It wasn't a proven premise that if you rewire a cell in vivo that you're going to get that same type of effect in vitro. This really demonstrates something that sounds feasible in retrospect – 'Of course you can rewire one and get the other' – but, that was not a known fact in the field whatsoever."

Focusing on carbon flux and future directions

The team reported producing BDO at the rate of 0.9 grams per Liter-hour in a system optimized for developing small-scale prototypes. Additionally, the team also used a similar rewiring strategy to produce the small molecules glycerol and itaconic acid to see how the cell-free system allowed them to increase the productivity rates for pathways of varying complexity. Rasor cautioned that further optimization is needed to scale up for commercial viability.

"For context, commercially relevant biobased processes in cells often have synthesis rates on the order of one to five grams per Liter per hour," Jewett said. "From the academic perspective we're able to be in this range in the absence of cell walls. If we can continue to learn how to focus the carbon flux, we can avoid transport limitations to potentially reach even higher productivity and that's one of these things that we're really excited about in the future, to see if that's possible."

Alper and Jewett noted this paper is the first result of the collaboration with the JGI, where they are partnering with Yasuo Yoshikuni and Ian Blaby of the DNA Synthesis Science Program. "This collaboration brings tremendous strengths from each of the groups to allow us to do research that, frankly, neither one of us would have done alone," Jewett said.

"To paraphrase a quote from an old movie, 'This is the start of a beautiful collaboration,'" Alper said. "We're exploring not just what can be enabled by this technology, but also what we can understand through this technology."

"This is a fantastic example of rapid-prototyping systems for metabolic pathways that we plan to offer to users in the near term," added JGI Director Nigel Mouncey.